Plant extract, compositions containing same, method of extraction and uses thereof

ABSTRACT

An extract from leaves of mulberry is disclosed. The extract has an IC 50  value sufficient to inhibit α-glucosidase I. The extract may comprise 5-40% (w/w) total imino sugars and 20-70% (w/w) total amino acids. The extract may reduce the production of melanin for the treatment of such ailments or diseases caused by pigmentation as freckle, chloasma, striae gravidarum, sensile plaque and melanoma. The extract may also control blood glucose level.

The present invention relates to a plant extract, compositionscontaining same, a method of extraction and to the use of said extract.

BACKGROUND

Mulberry leaves have a long history of medicinal use in Asian countries,in particular China. In recent years, phytochemists have isolated anumber of imino sugar constituents from mulberry leaves, such as1-deoxynojirimycin (DNJ), fagomine, and N-methyl-DNJ. The chemicalstructures of the imino sugars are similar to that of monosaccharides,being mostly polyhydroxyl heterocyclics with a 5 to 6-membered ring. Thekey difference between the two lies in the hetero atoms of theheterocycle. The former contains nitrogen atoms (N) while the latteroxygen (O).

It has been shown that the imino sugar constituents from mulberry leavesexhibit certain inhibitory activity on α-glucosidase I and II, amongwhich DNJ showed the strongest activity. Further pharmacologicalexperiments revealed that DNJ acted on melanocyte inhibiting thematuring process of TYR, which resulted in the reduction of melaninproduction. (Genji Imokawa, Analysis of Carbohydrate PropertiesEssential for Melanogenesis in TYRs of Cultured Malignant Melanoma Cellsby Differential Carbohydrate Processing Inhibition. The Journal ofInvestigative dermatology, 1990, 95(1):39-49; Ju Young Park, hyunjungChoi, Jae Sung hwang, Junoh Kim, Ih-Seop Chang, Enhanced depigmentingeffects of N-glycosylation inhibitors delivered by pH-sensitiveliposomes into HM3KO melanoma cells, Journal of Cosmetic Science, 2008,59:139-150.)

The inventors of the present invention carried out sets of enzymeexperiments and found that the total imino sugar extract (as measured bythe content of 1 deoxynojirimycin (DNJ), N-methyl-DNJ, and fagomine)from mulberry leaves as described in the extracts of the presentinvention possessed more potent inhibitory activity on α-glucosidase Iand II than the pure chemical DNJ. This discovery makes it possible touse such mulberry extracts to achieve inhibition of α-glucosidase I andII with lower concentrations of DNJ, thereby reducing the likelihood ofpossible adverse drug reaction (ADR) and making the finished productsafer to use. In addition, since the cost of producing the mulberryextract, as described in the present invention, is much lower than thatof obtaining the pure chemical DNJ, the cost of treatinghyper-pigmentation related ailments could be greatly reduced.

Thus, cosmetics and pharmaceuticals made from such extracts have hugeadvantages in efficacy, safety and cost over those containing purechemicals such as DNJ, the structure of which is given below.

Structure of 1-Deoxyjirimycin (DNJ)

In the past, there have been a number of researchers in China or abroadwho have tried to commercialise mulberry extracts in the beauty productmarket for whitening and spot-reduction. However, by comparison, thepresent extract has quite different characteristics and offers a numberof advantages.

Chinese patent ZL99123894X discloses a composition of plant extracts forthe treatment of skin pigmentation, which is composed of a combinationof three main ingredients, namely an extract from plants of the genusMorus, an extract from plants of the genus Scutellaria and derivativesof salicylic acid. The Morus extract of that invention was obtained frommulberry root, which contained mainly kuwanone and there were no clearspecification of the extract.

US patent application No. 347884 discloses the use of an extract fromthe branches of mulberry tree for the same cosmetic purposes, with theactive constituents in the extracts being oxyresvertrol andmulberroside. Compared with these two inventions, the present inventionhas the following advantages and features:

-   -   1. Raw material advantage. The raw material, mulberry leaves,        used in present invention, is easier to regenerate, and offers a        more sustainable resource than roots and branches. It also        offers a relatively lower cost than the two inventions mentioned        above.    -   2. Different mechanisms of action. In the two inventions        mentioned above, the activity was achieved through competitive        inhibition on TYR. In the present invention, the mechanism of        action is to reduce the production of melanin through inhibiting        α-glucosidase, resulting in less mature (and less/inactive) TYR.    -   3. Different active principles. In the above mentioned patents,        the active principles were either flavonoids, e.g. kuwanones, or        diphenyl ethenoids, e.g. oxyresveratol and mulberrosides. In        contrast the active principles of the present invention are        imino sugars, and different preparation methods are employed to        isolate an extract rich in these imino sugars.    -   4. Unique preparation process. The process is designed to ensure        optimum extraction of the active ingredients and purification        which to improve e.g. the physical properties of the extract,        making it more suitable to be used in, for example, cosmetic        products.

Melanin is the most important factor in determining the colour of humanskin. It is biosynthesized in the melanosome of melanocytes at the baselayer of the epidermis. Under normal physiological condition, melaninprotects the skin from UV light injury. When melanin synthesismetabolism is disturbed by external factors, hormone disorders, senileprocesses, etc., the melanin at the epidermis base layer will increaseand the colour of the skin will darken. This in turn will result inpigmentation ailments or diseases such as freckle, chloasma, striae ofpregnancy, senile plaque and melanoma. Besides, there are a great numberof beauty-conscious people who are longing for whiter skin and thusthere is a demand for skin lightening, whitening and spot reducingagents and cosmetics.

In brief, the biosynthesis of melanin includes the following steps

Tyrosinase (TYR), a type of glycoprotein containing ionic copper, and isthe key enzyme for the biosynthesis of melanin. It catalyses thereaction to transform tyrosine to dopa and dopaquinone. TYR isconsidered an important target for reducing pigmentation and is usedfrequently in the research area for products with the function ofwhitening skin and reducing spots.

Currently the main way of targeting TYR is to inhibit its formation andactivity to reduce the production of melanin. Current productscontaining TYR inhibitors include 1,4-dihydroxybenzene (hydroquinone)and its derivatives, kojic acid and its derivatives, and arbutin.

While 1,4-dihydroxybenzne and its derivatives are able to inhibit 100%activity of TYR, they also stimulate melanocytes and show cytotoxity.Prolonged use coupled with exposure to light could cause exogenouspigment spots. They are therefore banned in skincare products.

Kojic acid is very stable and has good effects in reducing pigment spotsthrough chelating copper ions to lower the activity of TYR. However,long term use of kojic acid could cause cytotoxicity resulting in skindiseases. Japanese researchers demonstrated that kojic acid could causeliver cancer (Tamotsu Takizawa, Toshio Imai, Jun-ichi Onose, MakotoUeda, Toru Tamura, Kunitoshi Mitsumori, Keisuke Izumi and Masao Hirose.Enhancement of Hepatocarcinogenesis by Kojic Acid in Rat Two-StageModels after Initiation with N-bis(2-hydroxypropyl)nitrosamine orN-diethylnitrosamine. Toxicological Sciences 2004 81(1):43-49).

Arbutin is considered as a whitening and spot-reducing beauty productwith very little side effects but it is highly light-sensitive and as aresult, large amounts of sun protection agents are required to be addedto the finished product, which increases the burden to the skin and thusaccelerates its senile process.

The above shortcomings restrict the application of existing products inthe market of beauty products for whitening skin and reducing spots.

TYR is a protein with a sugar chain (glycoprotein). Modern research inbiochemistry has revealed that in the process of its production (andmaturity) the original sugar chain must undergo a series ofmodifications in order to transform the newly produced TYR into matureTYR which has the normal biological functions. α-Glucosidase I and IIare the key enzymes in this process. α-Glucosidase I is mainlyresponsible for “cleaving” the glucose moiety with α-1,2 linkage at thefar end of the sugar chain while α-Glucosidase II will, in two steps,cleave the remaining two glucose moieties, linked by an α-1,3connection. (Mehta A, Zitzmann N, Rudd P M, Block T M, Dwek R A.α-Glucosidase inhibitors as potential broad based anti-viral agents,FEBS Letters, 1998, 430(1):17-22).

It is believed that when α-glucosidase I and II are inhibited, themodification of the sugar chain of the glycoprotein is retardedresulting in no production of mature TYR. With “immature TYR” theproduction of melanin is consequently reduced in proportion. (HiroyukiTakahashi, Peter G. Parsons, Rapid and reversible inhibition of TYRactivity by glucosidase inhibitors in human melanoma cells, The Journalof Investigative dermatology, 1992, 98(4):481-487.)

Therefore, it would be a highly feasible approach to reduce theproduction of melanin and in turn the skin pigmentation by inhibitingα-glucosidase I and II to minimize the formation of mature TYR, asillustrated in FIG. 4 which shows the proposed mechanism of action forinhibition of melanin synthesis. Essential modification of tyrosinase,catalysed by α-glucosidase, is inhibited by the Morus extract resultingin inactive tyrosinase formation.

BRIEF SUMMARY OF THE DISCLOSURE

In accordance with the present inventions there is provided a plantextract, obtained from Morus plant leaves, which has an IC 50 value toinhibit α-glucosidase I at a concentration of less than 90 μg/ml.

Preferably the plant has an IC 50 value to inhibit α-glucosidase I at aconcentration of less than 70 μg/ml and more preferably from 5-60 μg/mland more preferably still 5-40 μg/ml.

The IC₅₀ can be determined as set out in Experiment 1.

The improvement in the extracts activity over pure isolated DNJ isconsidered to be due to the activity of the other imino sugars presentin the extract. Preferably the extract comprises 5-40% (w/w) total iminosugars as measured by quantitative HPLC and/or LC-MS (liquidchromatography/mass spectrometry). The measured imino sugars include 1deoxynojirimycin (DNJ), N-methyl-DNJ, and fagomine.

More preferably the extract will comprise 8-30% (w/w) total iminosugars, more preferably still 15-20% (w/w) total imino sugars.

By accurately controlling the content of the characteristic chemicalconstituents, it becomes possible to effectively control the quality ofthe product thus providing certain assurance as to the cosmetic ortherapeutic effect of the product.

Most preferably the extract will further comprise the imino sugars1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 2-O-α-D-galactopyranosyl-DNJ(GAL-DNJ) and calystegin B.

The presence of DAB, which is a glycogen phosphorylase inhibitor, may beof significance in the extracts use in controlling sugar metabolismwhich application constitutes a further aspect of the invention.

In addition to the imino sugars the extract preferably comprises 20-70%(w/w) total amino acids.

More preferably the extract contains from 30-60% (w/w) total aminoacids, more preferably still 40-50% (w/w) total amino acids.

The amino acids present in the extract include 8 essential amino acidswhich human body is incapable of producing and include arginine,leucine, lysine and phenylanaline which promote the secretion ofinsulin. As it is well known, amino acids are essential for maintainingmoisture and elasticity of the skin. Amino acid deficiency will weakenthe skin metabolism and accelerate the skin senile process. (Marty J.P., NMF and cosmetology of cutaneous hydration. Annales de Dermatologieet de Vénéréologie, 2002, 129(1 Pt 2):131-136.). The extract of thepresent invention will thus also function to help maintain skin health.

The extract of the invention is obtained from Mulberry leaves of thefamily Moraceae, genus Morus and is selected from

-   -   a. Morus alba L.    -   b. Morus alba var. multicaulis L.    -   c. Morus nigra and    -   d. Morus australis Poir.

The inventors of the present invention carried out chemical analysis ofthe chemical constituents of the leaves of common Morus plants and foundthat the leaves of the above-mentioned species all contained relativelyhigher imino sugar content than other species and the leaves of M. albahad the highest content of imino sugars.

More preferably the imino sugar content is standardised with referenceto DNJ which is present in an amount of from 1-20% (w/w) of (DNJ)calculated on the basis of the total weight of the extract, morepreferably 2-10% (w/w) of (DNJ) calculated on the basis of the totalweight of the extract and, depending on the intended use, either 4-6%(w/w) of (DNJ) calculated on the basis of the total weight of theextract or 1-3% (w/w) of (DNJ) calculated on the basis of the totalweight of the extract. DNJ is also the predominant (highest yielding)imino sugar present.

The extract of the invention differs from current Mulberry extracts in anumber of ways including:

-   -   it's colour—it is pale yellow,    -   it's solubility—it is easily soluble in water,    -   its pH—it has a pH value of between 5.5-6.5 in a 1% water        solution, and    -   it's UV spectrum—it shows a maximum absorption at 218 nm and 263        nm.

The extract may be produced using a novel extraction and purificationprocess comprising three basic steps which process constitutes a furtheraspect of the invention.

In accordance with a second aspect of the present invention there isprovided a method for producing an extract, from Morus plant leaves,which has an IC 50 value to inhibit α-glucosidase I at a concentrationof less than 90 μg/ml comprising the steps of:

-   -   a. Conducting a water or alcoholic extraction step from leaf        material;    -   b. Conducting a column chromatography purification step using a        strong acidic cation exchange resin, washing with water, and        eluting with an ammonia solution, collecting the eluent, and        removing the ammonia therefrom;    -   c. Subjecting the eluent to column chromatography using a        macroporous absorption resin, collecting the solution; and    -   d. Concentrating and drying the extract.

Preferably the plant leaves were oven dried and made into coarse powderand

-   -   a. the extraction step is conducted with 5-18 folds of 0-40% low        molecular weight alcohol per the quantity of the Morus plant        leaves (w/w), and repeated up to 5 times,    -   b. the column is washed with 1-2 folds of water of the column        volume and the wash discarded, the column is eluted with 2-8        folds of 0.2-1.0N ammonia water solution of the column volume,        at an elution rate of 1-3 folds of the volume of the column per        hour, and the eluent collected with a pH value of between 9.0        and 11.0,    -   c. the column is eluted with a volume ratio between the solution        and column of between 20:1 to 5:1, and    -   d. the dried extract is pulverized to pass through a mesh 80        sieve.

Fuller details of the extraction process to obtain the extract of theinvention are set out below:

Extraction (Step 1):

The plant leaves were oven dried and made into a coarse powder. Extractthe powder with 5-18 folds of the quantity of the raw material (w/w) of0-40% low molecular weight alcohol for 1-5 times, preferably with 10-13folds of the quantity of the raw material (w/w) of 20-30% low molecularweight alcohol and more preferably with 11 folds of the quantity of theraw material (w/w) of 25% low molecular weight alcohol, to obtain theliquid extract.

Purification 1 (Step 2):

Column chromatography using a cationic exchange resin: After filtrationof the liquid extract obtained in step (1), the solution is put througha column filled with a strong acidic cationic resin. Elute the columnwith 1-2 folds, preferably 1.5 folds, of the column volume of water, anddiscard the eluent. Further elute the column with 2-8 folds of thecolumn volume of 0.2-1.0N ammonia water solution, preferably 5 folds,0.7N ammonia water solution, with an elution rate of 1-3 folds of thevolume of the column per hour, preferably 1.5 folds per hour. Collectthe eluent with pH values of between 9.0 and 11.0.

Purification (Step 3):

Column chromatography using macroporous absorption resin: Remove ammoniafrom the eluent in step (2) and adjust to pH 7. Put the solution throughthe column filled with a macroporous absorption resin. The volume ratiobetween the solution and column should be 20:1 to 5:1, preferably 15:1to 10:1 and more preferably 13:1. Concentrate the collected fluid runthrough the column and dry the concentrate, which is then pulverized topass through a mesh 80 sieve to obtain the extract of the presentinvention.

Based on the aforesaid extraction method at step (1), the said lowmolecular weight alcohol should be straight chain alkyl alcohol with nomore than 4 carbon atoms, preferably, methanol or ethanol. The saidextraction is reflux extracted for 1-3 hours, preferably 1-2 hours andmore preferably 2 hours.

After filtration of the liquid extract obtained in step (1), furthermeasures can be adapted to remove more impurities, improve thebioactivity of the extract and decolourize the extract. Those measuresinclude alcohol precipitation, flocculent precipitation, and othersappropriate for the removal of proteins, tannins and polysaccharides.The precipitates can be removed by either centrifuge or filtration. Thepreferred purification method herein is alcohol precipitation andcentrifuging: Concentrate the infusion to of the original volume, add1-3 folds of 95% ethanol, stir for half an hour and leave it for 8-12hours, centrifuging at 12000 r/min for 15 min. Keep the supernatant forfurther purification.

Based on the extraction method described herein, the total volume of theliquid extract at step (2) should be 2-20 folds of the cationic exchangeresin column, preferably 10-15 folds and more preferably 13 folds.

Under these conditions the active constituents can be effectivelyabsorbed by the cationic exchange resin which helps increase the contentof the active chemicals.

The strong acidic cationic resin used herein can be selected from thefollowing types: 001X7 (#732), Amberlite IR-120, Dowex-50, Lewatit-100,Zrolit 225 or Diaion SK-1, etc, with 001X7 (732) as the most preferreddue to its better absorption property to concentrate the activeconstituents and lower cost.

During the elution of the cation exchange resin column using ammoniawater, the pH value of the eluent increased gradually. Biological andchemical analysis of the eluent revealed that the content of fractionswith pH 9-11 showed the highest activity and hence only those fractionswith pH 9-11 were collected.

Based on the extraction method described herein, the flow rate at step(3) should be 1-4 folds, preferably 2 folds, of the volume of the columnper hour. This ensures the maximum absorption of coloured impurities bythe macroporous resin ensuring good decolourisation.

Four types of macroporous resins can be selected for the aforesaidcolumn chromatography: AB-8, HP20, S-8 and YWD03F4. After processcomparison and validation, it was found that resin S-8 produced the bestdecolourization result and thus Type S-8 resin is the preferredmacroporous resin material.

The mulberry leaf extract obtained according to the above process has amaximum absorption peak at 218.3 nm under UV scan and is pale yellow incolour, which is distinguishable from that of other mulberry leafextracts currently available on the market. Most mulberry leaf extractsused in beauty products on the market have deeper colour, i.e. eitheryellow or brownish yellow, resulting in coloured finished products,which is not considered as an ideal appearance.

The extract described in the present invention is easily soluble inwater and this provides for better diffusion and absorption of theactive ingredients on the targeted area. The mulberry extracts currentlyused in beauty whitening products contained flavonoids (e.g. kuwanones)and diphenyl ethenoids (e.g. oxyresveratol and mulberrosides) and as aresult they have relatively poor water solubility with averagediffusibility, which in practice requires the use of low molecularweight alcohol to improve solubility, thus increasing the burden on theskin.

The extract described in the present invention has a pH value of 5.5-6.5in a 1% water solution. This slightly acidic pH is close to the pH ofthe sebum film on the surface of the skin which is 4.5-6.6. When saidextract is made into skincare products, the irritation to the skincaused by the active constituents is therefore reduced.

The extract of the present invention may be formulated for use as apharmaceutical or cosmetic for use as a skin lightening agent or toreduce skin hyperpigmentation or as a pharmaceutical, nutraceutical orfood or drink ingredient to control blood glucose levels. Thus, it maybe used to, for example, treat type 2 diabetes or to lower the glycaemicindex of a food or drink.

In accordance with a third aspect of the present inventions there isprovided a plant extract according to the first aspect of the inventionor the product of the process according to a second aspect of theinvention for use as a cosmetic or medicament to treat conditions causedby pigmentation.

Preferably the cosmetic or therapeutic application is to reduce theproduction of melanin, including the treatment of ailments or diseasecaused by hyperpigmentation including: freckle, chloasma, striae ofpregnancy, senile plaque and melanoma.

A pharmacological experiment using the mulberry leaf extract of thepresent invention revealed that the said extract effectively inhibitedthe activity of α-glucosidase with higher potency than a quantitativelyequivalent sample of pure DNJ. It required only half the concentrationof DNJ for the extract to achieve the same effect to that of pure DNJ.When tested in cell lines, the said extract showed significantinhibition effect against melanin formation in melanoma cell lines A375and B16 and the potency was higher than that of frequently usedingredients in the marketed beauty whitening products, such as arbutinand magnesium L-ascorbyl-2-phosphate. The mechanisms of action of themulberry leaves extract of the present invention differ from that ofarbutin and magnesium L-ascorbyl-2-phosphate. The latter two directlyand competitively inhibit the activity of TYR, while the said mulberryleaf extract mainly inhibits the formation of the mature, active TYR.The advantage of said mulberry leaf extract includes higher potency andlonger action. Because of the different mechanisms of action, saidextract of the present invention can be used alone or together with theabove mentioned TYR inhibitors. A human clinical study confirms this invitro data and after 28 days of topical application of a 0.2% or a 0.5%mulberry leaf extract cream formulation there was a significantreduction in skin pigmentation (P<0.001) and a significant lightening ofthe skin (P<0.001)

In accordance with a forth aspect of the present inventions there isprovided a plant extract according to the first aspect of the inventionor the product of the process according to a second aspect of theinvention for use in controlling blood glucose level.

Preferably the extract for use in controlling blood glucose furthercomprises a glycogen phosphorylase inhibitor.

In the animal experiment using Wistar rat model, and the human clinicalstudy, the mulberry leaves extract of the present invention demonstratedsignificant blood glucose lowering effect.

The extract of the invention is particularly good since it benefits froma combination of actives. Thus, the imino sugar constituents, such asDNJ, inhibit α-glucosidase in the gut, resulting in the reduction ofabsorption of saccharides (Asano N., Glycosidase inhibitors: update andperspectives on practical use. Glycobiology, 2003, 13(10): 93R-104R.).Another of the imino sugars 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1),was found to have strong inhibitory activity against liver glycogenphosphorylase and also to inhibit in vivo the decomposition of liverglycogen. As a result it is anti-hyperglycaemic. Furthermore, thepresence of the amino acids such as arginine, leucine, lysine andphenylalanine, have the function of promoting the secretion of insulin.

Thus, the extracts described in the present invention can be use tocontrol after-meal blood glucose levels and adjust blood glucosebalance.

Such extracts can be formulated for use as a medicament or healthproduct (such as a food additive or supplement) for the control of bloodglucose.

To provide for the third aspect the extract may be formulated as acosmetic or medicament comprising excipients and optionally one or morealternative actives.

For use as a skin lightening agents other actives may be selected from,for example, vitamin C and its derivatives, such as vitamin-magnesiumphosphatidate, kojic acid, arbutin, diacetylboldin, azelaic acid,octadecenedioic acid, undecylenoylphenylalanine (DEP-11), licoriceextract, Aloe extract, watercress (Nastutium officinale) extract,Ascophyllum extract (Ascophyllum nodosum), hops (Humulus lupulus)extract, glutathione, ecdysone and/or ellagic acid.

A preferred cosmetic or medicament comprises the extract of theinvention together with the vitamin C derivative, magnesiumL-ascorbyl-2-phosphate (VC-PMG).

Preferably, in a combination product the extract is present in the ratioof 10:1 to 1:1 extract to other skin lightening agent, more preferably5:1.

When manufacturing cosmetics or drugs for skin diseases using themulberry leaf extract of the present invention or a composition alsocontaining other ingredients, all the common base materials acceptablein pharmaceutics can be used, which include water soluble base materialssuch as glycerin, polyethylene glycol, cellulose derivatives, etc andliposoluble base materials such as fat, lipids, Hydrocarbons, etc. Thefollowing excipients are also commonly used, and include preservatives,such as nipagins, chlorobutanol and sorbic acids; antioxydants such assodium sulphite, sodium bisulfite, BHT, etc; thickeners, such as stearicacid, bees wax, paraffin, laury alcohol, carboxmethyl cellulose (CMC),etc; emulsifiers such as triethanolamin, glycerol monostearate, Tweens,etc; sunscreen agents such as octyl methoxycinnamate, benzophenone-3,etc; humectants (moisturizer) such as glycerin, propylene glycol,sorbitol, etc; deodorants, fragrance and colouring agents, etc. Thequantities of the above mentioned excipients will be known to theskilled person.

When the said extract is used in cosmetic products for reducing theproduction of melanin, the recommended quantity in the finished productshould be 0.05-2%, preferably 0.1-1% and more preferably 0.2-0.5%(wt/wt).

To provide for the forth aspect the extract may be formulated in apharmaceutical or nutraceutical, added to a food or drink or provided asa supplement for adding thereto in an effective amount.

In a food or drink it functions to lower the glycaemic index. In suchcases a dose of from 50-600 mg per serving (depending on size) may beused.

When manufacturing an oral drugs for the control of blood glucose levelthe mulberry leaf extract of the present invention may be formulatedwith common excipients for oral drugs such as, disintegrating agents(e.g., Dry starch, Carboxymethyl starch sodium, L-HPC, Cross linked-PVP,etc); lubricants (e.g., Magnesium stearate, Talc powder, Sodiumbenzoate, Polyethylene glycol 4000, etc) and adhesives (e.g., CMC).

When the said extract is used for controlling blood glucose level, basedon the concentrations of the active constituents in the extract, therecommended dose is 25-600 mg each time, 3 times daily; preferably100-300 mg each time, 3 times daily; more preferably 50-150 mg eachtime, 3 times daily.

Conventionally, the following forms of products can be made forcosmetics or drugs for skin diseases. These forms include solutionscontaining water, water-alcohol or oil, gel/colloids containing water oroil, micro-emulsions, dilute or thick emulsions, loose or dense powders,dispersions with oil in water phase formed with the aid of microparticles such as polymer particles and capsules, and best of all, ionor non-ion lipid vesicles.

When manufacturing cosmetics or drugs for skin diseases using themulberry leaf extract or a composition they may appear in the following“dosage forms” with relative fluidity: cream, ointment, lotion, milkyliquid, emulsion fluid, mucilage, paste, foam, aerosol, and anhydroussolid preparation (e.g. stick-shaped).

When manufacturing the oral drugs using the mulberry leaves extract ofthe present invention to control blood glucose level, they can be madeinto commonly used oral dosage forms such as tablets, capsules andpowders.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 is an HPLC fingerprint of the plant extract of Example 1 Theupper trace is the standard HPLC of DNJ and the bottom trace the HPLCfingerprint of the plant extract of Example 1;

FIG. 2 is the UV spectrum of the plant extract of Example 1;

FIG. 3 is a UV spectrum of a prior art extract;

FIG. 4 is a schematic illustrating the mechanism of action behind thepigmentation reduction;

FIGS. 5a and b show the level of α-glucosidase inhibition by Morusextract (5 a) and DNJ (5 b);

FIG. 6 is the comparative effect of DNJ, arbutin and Morus extract ontyrosinase;

FIG. 7 is the comparative effect of DNJ, VC-PMG and Morus extract onmelanin synthesis inhibition;

FIGS. 8a, b and c show comparative cell toxicity of DNJ (a), VC-PMG (b)and Morus extract (c);

FIG. 9 is the glucose lowering effect of Morus extract in Wistar rats;

FIG. 10 is the glucose lowering effects of Morus extract in humans; and

FIGS. 11a and b are the skin lightening effects of Morus extract inhumans

DETAILED DESCRIPTION

The HPLC of FIG. 1 was obtained using a set up as follows:

-   -   Instrument: Waters (USA) HPLC apparatus w600-2420-717, Empower        data process system.    -   Column: Old Shodex Asahipak NH2P-50E (250×4.65 μm) column used        in reverse.    -   Reagents: Acetonitrile-water containing 6.5 nmol ammonium        acetate.    -   Column: temperature 40° C.    -   Flow rate: 1.0 ml/min    -   Detector: W2420 ELSD    -   Gain: 100; Drift tube temperature 50° C.; Sprayer heating level        60%    -   Mobile phase: Acetonitrile:Water (containing 6.5 nmol ammonium        acetate) (84:16)

The UV spectrum of FIG. 2 and FIG. 3 was obtained using:

-   -   Instrument: UNICO UV-2100 Spectrophotometer    -   Scan wavelength: 200-600 nm    -   Sample concentration: 1 mg/ml

The plant extract of the invention may be obtained by methodology asdescribed with reference to Examples 1 to 5 and using different mulberryleaves as illustrated with reference to Examples 6 to 9.

The resulting plant extract can be formulated for use as a cosmetic ormedicament or as a food or drink supplement or additive as illustratedwith reference to Examples 10 to 16.

The different activities of the extract are further illustrated by wayof Experiments 1 to 8.

Example 1

Pulverize 100 kg of dry mulberry leaves of the species Morus alba andextract under reflux 3 times (1 hour each time) with 12 folds of 30%ethanol (compared to the weight of the raw material). Condense theextract to a given volume and pass it through a column filled with astrong acidic cationic resin, such as 001X 7. The volume of the columnwas 1/14 of the extracted liquid. The column was washed with water (1.5folds of the column volume) and the component of interest (imino sugars)released from the column using 0.7 N ammonia water (5 folds of thecolumn volume), at a flow rate of 1.5 folds of the column volume perhour. The ammonia water eluent with a pH of 9-11 was collected. Theeluent was condensed to a given volume, the ammonium removed and the pHadjusted to pH 7. The eluent was then passed through a column filledwith a macroporous resin, such as S-8. The volume of the column was 1/10of the eluent liquid and the flow rate 2 folds of the column volume perhour. The collected fluid was then dried under vacuum and the driedproduct pulverized to pass through an 80 mesh sieve. 1.4 kg of a paleyellow powder was obtained, which contained 5.8% DNJ, 21% total iminosugars and 48% total amino acids.

The imino sugar content was determined using an assay as follows and theresult is shown in FIG. 1 the lower profile of which is a HPLCchromatogram showing the major imino peaks (the DNJ standard is shown inthe upper profile).

(a) Weigh accurately an appropriate quantity of DNJ as a control, addmethanol to make a solution of 0.1 mg/ml and shake well. Transfer Iml ofthe solution, measured accurately, to a 25 ml volumetric flask, to whichadd 1 ml of 0.4 mol/L boric acid-potassium chloride buffer solution (pH8.5) and 2 ml of 5 mmol/L FMOC-CI in anhydrous acetonitrile. Afterultrasonicating for 20 min. at room temperature, immediately add 2 ml of0.2 mol/L glycine solution and 0.1% acetic acid to volume, shake well toobtain the control sample solution.

(b) Dissolve 0.3 g of the extract in an appropriate amount of water andpass through a pre-treated polyamide column. Elute with a hydrochloricacid solution (pH 3). Collect the eluent, which was then condensed undervacuum to about 10 ml. Put the solution on to a pre-treated anionexchange resin column. Wash the column with water and collect theeluent. After being condensed under vacuum to about 30 ml, the eluentwas transferred to a 50 ml volumetric flask and water added to volume.Transfer 1 ml of the solution, measured accurately, to a 25 mlvolumetric flask and follow the above procedure in (a) starting from “towhich add 1 ml . . . ” to obtain the test solution.

(c) The solutions were run on an HPLC as follows and the imino contentdetermined therefrom (by Empower). Absorbent: C18-ODS; Mobile phase:acetonitrile:0.1% acetic acid (30:70), running for 30 min, change themobile phase to acetonitrile:0.1% acetic acid (70:30) running for 10min. Balance the system using the original mobile phase before injectingthe next sample. Chromatogram recording time was 30 min with thedetecting wavelength at 265 nm. Inject 20 μl, measured accurately, ofthe control solution and test solution, respectively, to the HPLCapparatus and run to obtain HPLC results.

The total amino acid content was assayed using a standard amino acidanalysis instrument.

Example 2

Pulverize 100 kg of dry mulberry leaves of the species Morus alba andextract under reflux 3 times (1 hour each time) with 18 folds of water(to the weight of the raw material). Pass the extract through a columnfilled with strong acidic cationic resin, such as Dowex-50. The volumeof the column was 1/20 of the extract. Wash the column with water (2folds of the column volume) followed by 0.5 N ammonium water (8 folds ofthe column volume) at a flow rate of 3 folds of the column volume perhour. Collect the ammonium water eluent with a pH of 9-11. Condense to agiven volume, remove the ammonium and adjust to pH 7. Run the eluentthrough a column filled with a macroporous resin, such as, HP20. Thevolume of the column was 1/20 of the eluent and the flow rate 4 folds ofthe column volume per hour. The collected fluid was then dried undervacuum and the dried product pulverized to pass through a 80 mesh sieve.1.4 kg of a pale yellow powder was obtained, which contained 4.3% DNJ,19% total imino sugars and 40% total amino acids.

The UV spectrum is illustrated in FIG. 2. The two distinct peaksdifferentiate the extract from other Morus extracts (See FIG. 3) whichcontain higher proportion of impurities. These impurities results in nodistinction between peaks, and could lead to lower levels of efficacy

Example 3

Pulverize 20 kg of dry mulberry leaves of the species Morus alba andextract under reflux 3 times with 5 folds of 40% ethanol to the weightof the raw material. Filter the combined extract, to which an equalvolume of ethanol was added, stir with constant speed for half an hourand leave it overnight. Remove the precipitate by centrifuging at12000r/min for 15 min, recover ethanol to a given volume. Pass thesupernatant liquid through a column filled with a strong acidic cationicresin, e.g.001X7 type. The volume of the column was ⅓ of the liquid.Wash the column with water (same amount as the column volume) and elutewith 1.0 N ammonia water (2 folds of the column volume) with a flow rateof the column volume per hour. Collect the ammonia water eluent with apH 9-11. Condense to a given volume, remove ammonium and adjust to pH 7.Pass the eluent through a column filled with a macroporous resin, suchas S-8. The volume of the column was 1/13 of the eluent and the flowrate, the column volume per hour. The collected fluid was then driedunder vacuum and the dried product pulverized to pass through an 80 meshsieve. 0.11 kg of a nearly white powder was obtained, which contained9.1% DNJ, 29% total imino sugars and 32% total amino acids.

Example 4

Pulverize 50 kg of dry mulberry leaves of the species Morus alba andextract under reflux 3 times with 12 folds of 40% ethanol to the weightof the raw material. Filter the combined extract and add a precipitationagent, aluminium potassium sulphate, to pH 3, and stir for 30 min. Afterleaving it for 2 hours, remove the precipitate by centrifuging. Adjustthe supernatant to pH 7. Pass the supernatant through a column filledwith a cationic resin, 001X7. The volume of the column was 1/13 of theliquid. Wash the column with water (2 folds of the column volume) andelute with 0.2 N ammonia water (8 folds of the column volume), at a flowrate of the column volume per hour. Collect the ammonium water eluentwith a pH of 9-11. Condense to a given volume, remove ammonium andadjust to pH 7. Put the eluent liquid through a column filled with amacroporous resin AB-8. The volume of the column was 1/20 of the eluentand the flow rate, 2 folds of the column volume per hour. The collectedfluid was then dried under vacuum, the dried product pulverized andpassed through an 80 mesh sieve. 0.12 kg of a nearly white powder wasobtained, which contained 18.6% DNJ, 39% total imino sugars and 21%total amino acids.

Example 5

Pulverize 50 kg of dry mulberry leaves of the species Morus alba andextract under reflux 4 times with 11 folds of 25% ethanol to the weightof the raw material. Filter the combined extract, which was then putthrough a cationic resin column filled with Amberlite IR-120 (H+) type.The volume of the column was 1/15 of the liquid. Wash the column withwater (1.5 folds of the column volume) and elute with 0.7 N ammoniawater (5 folds of the column volume), at a flow rate of 1.5 folds of thecolumn volume per hour. Collect the ammonia water eluent with a pH of9-11. Condense to a given volume, remove the ammonium and adjust to pH7. Put the eluent through a column filled with an S-8 macroporous resin.The volume of the column was ⅕ of the eluent and the flow rate was acolumn volume per hour. The collected fluid was then dried under vacuumand the dried product pulverized to pass through a 80 mesh sieve. 0.46kg of a pale yellow powder was obtained, which contained 5.6% DNJ, 24%total imino sugars and 48% total amino acids.

Example 6

Pulverize 5 kg of dry mulberry leaves of the species Morus alba var.multicaulis L. and extract under reflux 3 times with 11 folds of 25%ethanol to the weight of the raw material. Filter the combined extract,which was then put through a column filled with a cationic exchangeresin type 001X7. The volume of the column was 1/13 of the liquid. Elutethe column with water (2 folds of the column volume) followed by 0.5 Nammonia water (5 folds of the column volume) with a flow rate of 1.5folds of the column volume per hour. Collect the ammonia water eluentwith a pH of 9-11. Condense to a given volume, remove ammonium andadjust to pH 7. Put the eluent through a column filled with a macropousresin HP20. The volume of the column was 1/10 of the eluent liquid andthe flow rate 4 folds of the column volume per hour. The collected fluidwas then dried under vacuum and the dried product pulverized to passthrough an 80 mesh sieve. 48 g of a pale yellow powder was obtained,which contained 2.1% DNJ, 8.3% total imino sugars and 65% total aminoacids.

Example 7

Pulverize 5 kg of dry mulberry leaves of the species Morus nigra andextract under reflux 3 times with 8 folds of 80% ethanol to the weightof the raw material. Filter the combined extract and condense it toabout 6 L, to which about 12 L of ethanol was added. Stir with aconstant speed for half an hour and leave it overnight. Remove theprecipitate by centrifuging at 12000r/min for 15 min, recover theethanol to a given volume and add an appropriate quantity of aflocculant, such as aluminium sulphate. After complete precipitation, bysetting aside, remove the precipitate by centrifugation. Pass thesupernatant liquid through a column filled with a cationic exchangeresin, such as 001X7. The volume of the column was 1/10 of the liquid.Wash the column with water (same amount as the column volume) followedby 0.5N ammonia water (4 folds of the column volume) with a flow rate of3 folds of the column volume per hour. Collect the ammonia water eluentwith a pH of 9-11. Condense to a defined volume, remove ammonium andadjust to pH 7. Put the eluent liquid through a column filled with amacroporous resin HP20. The volume of the column was 1/15 of the eluentliquid and the flow rate, 4 folds of the column volume per hour. Thecollected fluid was then dried under vacuum and the dried productpulverized to pass through an 80 mesh sieve. 40 g of a nearly whitepowder was obtained, which contained 3.9% DNJ, 15% total imino sugarsand 58% total amino acids.

Example 8

Pulverize 5 kg of dry mulberry leaves of the species Morus australisPoir. and extract under reflux 3 times with 8 folds of 80% ethanol ofthe weight of the raw material. Filter the combined extract, which wasthen put through a column filled with Amberlite IR-120 (H+) typecationic resin. The volume of the column was 1/10 of the liquid. Elutethe column with water (1.5 folds of the column volume) followed by 0.7 Nammonia water (5 folds of the column volume) at a flow rate of 1.5 foldsof the column volume per hour. Collect the ammonia water eluent with apH 9-11. Condense to a given volume, remove ammonium and adjust to pH 7.Put the eluent liquid through a column filled with an AB-8 macroporousresin. The volume of the column was 1/13 of the eluent liquid and theflow rate, 2 folds of the column volume per hour. The collected fluidwas then dried under vacuum and the dried product pulverized to passthrough an 80 mesh sieve. 42 g of a pale yellow powder was obtainedwhich contained 1.4% DNJ, 5.2% total imino sugars and 68% total aminoacids.

Example 9

Pulverize 50 kg of dry mulberry leaves of the species Morus alba andextract under reflux once with 10 folds of 30% ethanol of the weight ofthe raw material. Filter the extract, which was then put through acolumn filled with Amberlite IR-120 (H+) type cationic resin. The volumeof the column was 1/13 of the liquid. Elute the column with water (1.5folds of the column volume) followed by 0.7N ammonia water (7 folds ofthe column volume) with a flow rate of 2 folds of the column volume perhour. Collect the ammonia water eluent with a pH of 9-11. Condense to agiven volume, remove ammonium and adjust to pH 7. Put the eluent througha column filled with an AB-8 macroporous resin. The volume of the columnwas 1/15 of the eluent and the flow rate was the column volume per hour.The collected fluid was then dried under vacuum and the dried productpulverized to pass through an 80 mesh sieve. 0.5 kg of a pale yellowpowder was obtained which contained 4.2% DNJ, 22% total imino sugars and46% total amino acids.

Example 10

Mix 8 portions of stearic acid, 5 portions of glycerol monostearate, 3portions of liquid paraffin, 8 portions of spermaceti wax, 1 portion ofbee wax and 5 portions of silicon oil, and heat to 75° C. (forming “Base1”). Mix 0.7 portions of triethanolamine, 5 portions of glycerin, 0.05portions of methylparaben and heat to 75° C. (forming “Base 2”).). Tothe combined bases 1 and 2, add 3 portions (w/w) of the mulberry leafextract of Example 1, mix well and add water to 100 ml. When cooled addan adequate amount of fragrance to produce a beauty whitening creamcontaining the said mulberry leaf extract.

Example 11

Mix 7 portions (w/w) of glycerine, 4 portions of propylene glycol, 0.2portions of 30% NaOH and 0.1 portions of potassium sorbate. Adddistilled water to 100 ml to obtain the water phase. Separately mix 10portions of stearic acid, 8 portions of butyl stearate, 1 portion ofglycerol monostearate and 3 portions of stearyl alcohol and heat toobtain the oil phase. Heat the above water phase to 95° C. Slowly addthe oil phase to the water phase at the same temperature with continuousstirring. At about 45° C., add to the mixture 2 portions of the mulberryleaf extract of Example 4 and 0.4 portions of magnesiumL-ascorbyl-2-phosphate, and a few drops of fragrance, and keep stirringuntil the two phases are mixed. Cool to obtain a paste.

Example 12

Mix 7 portions (w/w) of glycerine, 4 portions of propylene glycol, 0.2portions of 30% NaOH and 0.1 portions of potassium sorbate. Adddistilled water to 100 ml to obtain the water phase. Separately mix 10portions of stearic acid, 8 portions of butyl stearate, 1 portions ofglycerol monostearate and 3 portions of stearyl alcohol and heat toobtain the oil phase. Heat the above water phase to 95° C. Slowly addthe oil phase to the water phase at the same temperature with continuousstirring. At about 60° C., add to the mixture 2 portions of the mulberryleaf extract of Example 1 and 0.2 portion of DEP-11. Add a few drops offragrance and keep stirring until the two phases were mixed well. Coolto obtain a paste.

Example 13

To 0.4 portions (w/w) of the mulberry leaves extract of Example 5 add0.1 portions of ethylparaben, 0.5 portions of sodium bisulphate, 0.1portions of disodium edentate and 9 portions of glycerine, and adddistilled water to 100 ml to obtain the lotion.

Example 14

Mix well 5 kg of the mulberry leaf extract of Example 1 with 1.8 kg ofstarch, 1.5 kg of microcrystalline cellulose, 0.45 kg of cross-linkedPVP, 0.55 kg of CSM-Na, and appropriate quantities of magnesium stearateand silicon micro-powder. Make 20,000 tablets, weighing about 0.5 geach.

Example 15

Mix 1.0 kg of the mulberry leaf extract in Example 3 with an appropriatequantity of starch, fill in 10,000 capsules to make each capsule contain100 mg of the extract.

Example 16

To 2.0 kg of the mulberry leaf extract of Example 3 add 0.5 kg ofvitamin C, 1.0 kg of citric acid, 0.8 kg of sodium bicarbonate, 0.08 kgof mannitol, PVP, PEG 6000, flavouring agent and bonding agent to makeeffervescent granules.

Activity Test Experiments Experiment 1. Inhibition Activity Assay of theMulberry Leaf Extract on α-Glucosidase

The aim was to investigate the inhibition activity of the mulberryleaves extract in the present invention on α-glucosidase. The reagentsand apparatus are set out below:

-   -   (1) Mulberry leaves extract of Example 1,    -   (2) DNJ standard reference chemical,    -   (3) α-glucosidase (Type I from Bakers yeast, EC232.604.7), made        into a 0.42 U/ml solution using 0.1 mol/L phosphoric acid buffer        (pH 6.8)    -   (4) pNPG, made into a 5 mmol/L solution using 0.1 mol/L        phosphoric acid buffer    -   (5) pNP, made into a 200 μmol/L solution using 0.1 mol/L        phosphoric acid buffer (pH 6.8)    -   (6) Multiskan Ascent microplate reader (Thermo Electron Co.,        USA)

The method was as set out below:

Standard Curve

Dilute the 200 μmol/L pNP solution using 0.1 mol/L phosphoric acidbuffer, respectively, to 100 μmol/L, 50 μmol/L, 25 μmol/L, 12.5 μmol/L,6.25 μmol/L and 3.125 μmol/L. From each of the diluted solutions take200 μl for OD measurements at 405 nm and use the OD values to draw thestandard curve.

Test Sample Assay

-   -   (1) Put 80 μl each of the test sample of different        concentrations into individual wells of the microplate and 80 μl        phosphoric acid buffer solution as placebo.    -   (2) To each well, add 30 μl enzyme (0.42 U/ml), place on the        bench for 30 s, incubate together with the substrate (pNPG) for        15 min. at 37° C. and turn on the microplate reader, set the        temperature at 37° C., measurement mode to kinetic and interval        at 10 s. Take the readings 13 times (total 2 min.).    -   (3) To each well add 90 μl substrate (5 mmol/L pNPG) shake on        the bench for 30 s, place into the microplate reader, press        START, continuously measure the OD values at 37° C.    -   (4) With the concentrations of pNP as X axis and OD values as Y        axis, draw the standard curve. Use the OD values obtained in        step (3) against the standard curve to get related quantities of        the reaction product.    -   (5) For the placebo and the test samples of different        concentrations, take the time as X axis and product quantity as        Y axis to draw a reaction progress curve. The slope of the        straight line is the reaction speed.    -   (6) With the concentration of the test samples as X axis and        reaction speed as Y axis, establish the test sample        concentration curve and obtain IC₅₀. Inhibition activity        (U/μg)=(0.5× enzyme activity of each well)/(IC₅₀×the volume of        the samples of each        well)=(0.5×0.42×0.03)/(IC₅₀×0.08)=0.07875/IC₅₀.

Results and Discussion:

The results of the above experiment showed that the IC₅₀ of the mulberryleaves extract in Example 1 had an IC₅₀ value of 13.6 μg/ml ininhibiting α-glucosidase whilst that of pure DNJ was 70 μg/ml. Thisdemonstrated that the activity of the former was much greater than thelatter. Considering the fact that the DNJ content in the mulberry leavesextract in Example 1 was only 3.5%, it becomes possible to use the saidextract to achieve the same or better activity whilst reducing thelikely adverse reactions caused by the use of higher concentrations ofthe pure compound. See FIGS. 5a and b . which show respectively the invitro inhibition of α-gucosidase by Morus extract and DNJ, Initialreaction velocity versus concentration.

Experiment 2: Inhibition Activity Assay of the Mulberry Leaf Extract andArbutin on TYR

The aim was to investigate the impact and mechanisms of action of thetest samples on the TYR activity in melanoma cell line B16. The reagentsand apparatus were as set out below:

-   -   (1) Mulberry leaf extract of Example 1    -   (2) DNJ standard reference chemical    -   (3) Arbutin standard reference chemical    -   (4) L-dopa solution 1 mg/ml in pH 6.8 phosphoric acid buffer,        prepared immediately before use    -   (5) WIP cell lysis buffer    -   (6) Cell culture plate (24 wells and 96 wells)    -   (7) Optical microscopy    -   (8) Centrifuge    -   (9) Microplate reader        The methods were set out as below

B16 melanoma cells were washed with phosphate-buffered saline andcollected in a centrifuge tube. After centrifugation, WIP-lysis buffercontaining phenyl-methylsulfonyl fluoride (PMSF) was added to the cellpellet to lyse the cells and the solution was left to stand on ice for30 minutes. The solution was subsequently centrifuged for 10 min, at12000 g, at 4° C., with the supernatant (cell extract) containing thecellular components being retained for assay purposes.

To determine the direct effect of DNJ, arbutin, and Morus extract ontyrosinase catalytic activity, 60 μl DNJ, arbutin or Morus extract(concentration of compounds used varied) were added to 60 μl cellextract in a 96-well microplate. The plate was incubated at 37° C. for 1hour, and then 80 μl L-dopa was added to each well. The absorbance (492nm) of each well was recorded at 5 minute intervals for 30 minutes at37° C. using a microplate reader.

To determine the indirect effect of DNJ, arbutin, and Morus extract ontyrosinase catalytic activity, B16 melanoma cells were cultured withdifferent concentrations of DNJ, arbutin, or Morus extract for threedays prior to extraction and assessment. Following extraction, 40 μlcell extract, 120 μl phosphate buffer (pH 6.8), and 40 μl of L-Dopa (2mg/ml) were placed in a 96-well microplate, and the absorbance of eachwell at 492 nm was recorded at 5 minute intervals for 30 minutes at 37°C. using a microplate reader.

Results and Discussion

The results of the assays can be seen in FIG. 6. The light columns showthe direct effect of DNJ, arbutin, and Morus extract on tyrosinaseactivity in the cell extracts, whilst the dark columns show the resultsof the indirect effects of the compounds of tyrosinase activity.

In the direct activity assay, arbutin reduced the activity of tyrosinasein a dose-dependent manner, whilst DNJ and Morus extract showed noeffect on tyrosinase activity. However, in the indirect activity assay,after the cells had been incubated for three days incubation in thepresence of the test compounds, there was a dose-dependent decrease intyrosinase activity in the DNJ and Morus extract groups, but cellsincubated with arbutin showed no effect on tyrosinase activity.

These findings are in-line with the reported literature and support theproposed different mechanism of action for the α-glucosidase inhibitors,DNJ and Morus extract. In the direct activity assay, arbutin directlyinteracted with tyrosinase, binding to the active site and inactivatingthe enzyme, therefore reducing the oxidation of L-dopa to o-dopaquinone.α-Glucosidase inhibitors do not directly affect the activity oftyrosinase and this was reflected in the results of the direct activityassay where DNJ and Morus extract were shown to have no tyrosinaseinhibitory activity.

In the indirect activity assay, both DNJ and Morus extract were shown tohave inhibitory activity over tyrosinase. α-Glucosidase inhibitors acton immature tyrosinase, and by preventing the binding of calnexin duringtyrosinase maturation, there are conformational changes that affect theenzymatic activity of the mature tyrosinase. These changes wereevidenced by the decrease in enzymatic activity after three daysincubation with DNJ and Morus extract. Arbutin directly competes for thetyrosinase active site and in the indirect assay, where arbutin was notpresent in the assay solution, there was no inhibition of enzymaticactivity.

Experiment 3. Impact on the Melanin Content in Melanocytes by theMulberry Leaf Extract of the Invention

The aim was to investigate the impact of the test samples on melanincontent of melanoma cell line B16. The reagent and apparatus used areset out below:

-   -   (1) Mulberry leaf extract of Example 1    -   (2) DNJ standard reference chemical    -   (3) VC PMGstandard reference chemical    -   (4) WIP lysis buffer    -   (5) Optical microscope    -   (6) Cell culture plate (24 wells and 96 wells)    -   (7) Centrifuge    -   (8) Microplate reader

The method was as set out below:

-   -   1. Harvest the melanoma B16 cells at Log phase and adjust the        cell suspension to an appropriate concentration, divide into 24        well plates and allow the cells to adhere to the walls.    -   2. To each well add 1 ml test sample of different concentration        made by diluting with the medium (4 duplicates for each        concentration). Incubate for 2 days, refresh the sample solution        and continue to incubate for 2 days.    -   3. Harvest the cells on day 4 and wash thoroughly twice with        PBS. Add 250 μl WIP lysis buffer and allow to lyse in an ice        bath, vortex mixing once every 5 min for 4-5 times.    -   4. Centrifuge for 10 min at 12000 rpm at 4° C. Discard the        supernatant and dissolve the precipitate with 400 μl 1N NaOH and        keep it in a 80° C. water bath for 1 hour.    -   5. Use a microplate reader to measure OD value at 405 nm, and        calculate the relative content.        Results and discussion:

The results of the assays can be seen in FIG. 7. The midtone columnsshowed the inhibitory effect of DNJ on melanin content in B16 melanomacells, whilst the dark columns and light columns showed the results ofthe inhibitory effects of VC-PMG and Morus extract on melanin content inB16 melanoma cells. All effects are after 3 days incubation with DNJ,VC-PMG or Morus extract at different concentration.

Compared to the control the content of melanin in the treatments of DNJ,VC-PMG and Example 1 at different concentration levels decreasedsignificantly in a dose-dependent manner. The activity intensity of thethree compounds, from strong to weak, was: DNJ>Morus extract >VC-PMG.

DNJ is an active compound identified from Morus extract, as anα-glucosidase inhibitor andcan reduce the activity of tyrosinaseresulting in reduction of melanin in B-16 melanoma cell lines. Since thecontent of natural occurring DNJ in all plants is very low, it isimpossible to use natural DNJ for a commercial purpose. But with Morusextract, although the DNJ content in Morus extract is as low as 2-5%,Morus extract still demonstrated a similar potency of activity to DNJ,which indicated that within Morus extract, DNJ may work synergisticallywith other active compounds to generate a strong anti-pigmentationeffect.

From our observation, when the concentration of VC-PMG applied to theB16 melanoma cell increased to over 200 ppm, the viability of the celldecreased, which indicated that an excessive concentration of VC-PMG maycause harm to the cells. Morus extract, as a natural product, has abetter safety profile and a stronger efficacy compared to VC-PMG, andits mechanism of action differ from VC-PMG, thus Morus extract can beeither used alone or combined with VC-PMG to achieve a betteranti-pigmentation effect.

In order to develop Morus extract as the lightening products incosmetics, we considered the dosage of lightening ingredient usuallyused in cosmetics: arbutin: 1%-5%, kojic acid: 0.2%-3%, vitaminderivates: <3%. Based on our study, the recommend dosage of Example 1 isbelow 3%.

Experiment 4. Impact of the Mulberry Leaf Extract of the PresentInvention on the Growth of Melanocytes Methods Cell Viability

Cell survival was measured using the MTT assay, which is based on theconversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide to MTT-formazan crystal by mitochondrial enzyme in viable cells.MTT was freshly prepared at 2 mg/ml in phosphate-buffered saline (PBS).2×10⁴ cells per ml were plated in 96-well plates, DNJ, VC-PMG, Morusextract or DMEM were added to the culture medium. Aliquots of 20 μl ofMTT stock solution were added to the well at different time pointsthroughout the experiment, and the plate was incubated at 37° C. for 4 hin a humidified 5% CO2 incubator. After 4 h, the formazan crystals ofthese cells were solubilized in 100 μl DMSO by gentle shaking. After 10min, the amount of formazan was quantified spectrophotometrically usingan ELISA plate reader at 540 nm.

Determination of Melanin Content in Melanoma Cells

The B16 melanoma cells were seeded at a density of 4×104 cell per wellof 24-well culture plates, incubated at 37° C. under 5% CO2 atmospherefor 24 h. The cells were then treated with various concentrations ofDNJ, VC-PMG or Morus extract for 3 days. The cells were washed withphosphate-buffered saline, collected in centrifuge tube. Aftercentrifugation, cell pellets were disrupted in WIP lysis buffercontaining phenylmethylsulfonyl fluoride (PMSF) on ice for 30 min. Thesolution was centrifuged for 10 min at 12000 g at 4° C., the pelletswere dissolved in 1N NaOH by boiling for 1 h at 70° C. The melanincontent was assayed at 405 nm in a spectrophotometer.

Results and Discussion The Effects of DNJ, VC-PMG and Morus Extract onCell Viability

The data on the cell viability assay using MTT for B16 melanoma cellswere given in FIG. 8A, FIG. 8B and FIG. 8C.

DNJ, VC-PMG and Morus extract induced no growth inhibition of B16melanoma cells at the given concentration. These data clearly showed thenon-cytotoxic nature of DNJ, VC-PMG and Morus extract in B16 melanomacell below the concentration of 200 ppm, and indicate that cell growthwas not an influence for the assay of melanin content below.

Experiment 5: Impact of the Mulberry Leaf Extract of the Invention onthe Blood Glucose of Wistar Rats

The reagents and apparatus are set out below:

-   -   (1) Mulberry leaf extract of Example 1    -   (2) Saline    -   (3) Miglitol standard reference chemical    -   (4) 64 Wistar rats    -   (5) Johnson's One Touch Ultra blood glucose meter and Code 9        blood glucose test strips        The method used was as set out below:

Animals received Morus extract via intraperitoneal injection (15 mg/kgor 30 mg/kg) or intragastrically (25 mg/kg or 50 mg/kg). Miglitol(Glyset®) (25 mg/kg), an α-glucosidase inhibitor approved for type-2diabetes was used as a positive control. Fasted blood-sugar levels wererecorded as a baseline, and blood sugar levels were measured at timeintervals 05.hr, 1 hr and 2 hrs after receiving a starch meal. Theanimals were dosed with Morus extract prior to receiving the starchmeal.

Results and Discussion:

Blood glucose in 0.5 h,1 h,2 h after giving starch (X±SD, n=10)

Fasted Dose Glucose 0.5 h 1 h 2 h Groups (mg/kg) (mmol/L) (mmol/L)(mmol/L) (mmol/L) Normal / 3.07 ± 0.79 3.06 ± 0.77  3.51 ± 0.76 3.22 ±0.47  control ig 25 mg/kg 3.02 ± 0.71 2.38 ± 0.49*  3.20 ± 0.56 3.05 ±0.59  ig 50 mg/kg 3.02 ± 0.73 2.23 ± 0.40** 2.89 ± 0.59  2.7 ± 0.59* ip15 mg/kg 3.05 ± 0.80 2.61 ± 0.54  3.29 ± 0.39 3.15 ± 0.50  ip 30 mg/kg3.06 ± 0.78 2.34 ± 0.41*  3.43 ± 0.54 2.97 ± 0.41  Positive 25 mg/kg3.04 ± 0.74 2.27 ± 0.71** 2.98 ± 0.52 2.69 ± 0.35* Control{circumflexover ( )}

The results of showed that Morus extract significantly reducedblood-glucose levels. The intragastric route of administration showedthat the active compounds have good oral bioavailability and are notinactivated through metabolism. The both intragastic and intraperitonealdoses significantly lowered blood glucose levels with the 50 mg/kg dosebeing as effective as miglitol (Glyset®) in this study. See FIG. 9

Experiment 6: Effect of Mulberry Extract on Reducing Blood GlucoseLevels in Humans Methods

In a small human study, participants received either 400 mg Morusextract or placebo, plus 50 g soft sugar after an overnight fast. Bloodsugar levels were monitored for three hours. Additionally, one patientreceived 50 g miglitol to act as a positive control.

Results and Discussion

The data showed that Morus extract dramatically reduced blood sugarlevels, reducing the initial blood sugar spike by 59.91%, reducing theblood sugar levels over two hours by 50.78%, and over three hours by47.18%. Morus extract was able to reduce the glycaemic index of the softsugar from 83.8 to 41.2. See FIG. 10

Experiment 7: Effect of Mulberry Extract on Skin Lightening in HumansMethods

Mulberry extract as in Example 1 was made into 0.2% and 0.5% creamformulation for the study. Twenty female subjects (age 18 and older)having given informed consent, applied each formulation to predefinedareas on each forearm twice daily. Prior to first treatment,measurements of skin colour where measured using a Chromameter CR300 onthe defined zones.

After 28 days of treatment, subjects returned to the testing laboratorywhere new measurements of skin colour were made on the same definedzones as day 0.

Evaluations were made on three study parameters which were:

-   -   L* (from dark to light). This is lightness parameter of the        skin. An increase in this parameter characterizes a lightening        of the skin.    -   b* (from the blue to yellow). A decrease in this parameter        characterizes a decrease in the yellow constituent of the skin.    -   ITA° (Individual Typological Angle). This parameter shows the        skin pigmentation degree of a subject using the lightness (L*)        and cutaneous melanin parameters (b*). An increase in the ITA°        characterizes a decrease in skin pigmentation.

Results and Discussion

L* parameter increased significantly (P<0.001) after 28 days of dailyuse of the 0.2% product (+0.54 A.U. on average) representing anincreased lightness in the skin colouration. This effect was observed in90% of the subjects. See FIG. 11A

The individual typological angle (ITA°) parameter increasedsignificantly (P<0.001) after 28 days of daily use of the 0.2% product(+1 A.U. on average) indicating a decrease in skin pigmentation. Thiseffect was observed in 57% of the subjects. See FIG. 11B

For the 0.5% product: L* parameter increased significantly (P<0.001)after 28 days of daily use indicating an increase in skin lightness.This effect was observed in 86% of the subjects. See FIG. 11A

The individual typological angle (ITA°) parameter for the 0.5%formulation increased significantly (P<0.001) after 28 days of daily useof product indicating a decrease in skin pigmentation. This effect wasobserved in 67% of the subjects. See FIG. 11B

Experiment 8. Extract Data

The extracts of the present invention (Samples 1 to 3) differ from priorart extracts in their characteristics as illustrated in Table 1 below:

TABLE 1 IC₅₀ (μg/ml) on Imino sugar Water Sample α-glucosidase I contentColour solubility 1 34 12% Pale yellow Easily 2 42 19% Pale yellowEasily 3 21 29% Nearly white Easily 4 240  2% Yellowish brown Average 5180  5% Yellow Soluble 6 204  3% Brownish green Average 7 110 12%Brownish green Soluble 8 150  6% Brownish yellow Average

Explanation to Table 1 above:

Samples 1-3 refer to the products made in Examples 1-3;

Sample 4 refers to the product made according to patent Ser. No.03/139,028.5;

Sample 5 was made with the method described below: Pulverize mulberryleaves and extract under reflux with 80% ethanol for 3 times, 1-2 hourseach time. For the first time, use 6 folds (w/w) of ethanol of theweight of the raw material, followed by 4 folds for both 2^(nd) and3^(rd) times. Combine the extracts and leave it for 24 hours beforefiltration. Concentrate the filtrate solution and extract the condensedextract with water 5 times at 70-80° C. Each time use 10 folds of waterof the weight of the starting material (mulberry leaves). Adjust the pHof the combined water extract to pH 3-4 and add NaCl to 3-5%concentration. The liquid was subjected to D101 macroporous resinchromatography with a column of 60 cm (H)×3 cm (D). Wash with water tillthe eluent became clear and then elute with 5 folds of 30-50% ethanol ofthe column volume, flow rate 20/ml. Collect the eluent, condense and dryit to obtain the sample (5).

Sample 6 was made with the method described below: Pulverize mulberryleaves and extract twice with 50-60% ethanol at 50° C., using 10-12folds of ethanol of the weight of the mulberry leaves each time.Concentrate the extract and pass it through a column filled with D72strong acidic macroporous resin and collect the fluid (fraction 1).Elute with ethanol to obtain fraction 2 followed by 70% ethanolcontaining 2% ammonium to obtain fraction 3. Centrifuge and concentratefractions 1, 2 and 3, respectively. Dry and mix to obtain the sample(6).

Sample 7 was made with the method described below: Pulverize mulberryleaves and extract with 80° C. water twice, (10-15 folds of water).Adjust the pH of the combined extract to pH 2-3, cool it to 0° C. untilcomplete precipitation. Filter and subject the concentrated filtrate tocationic resin chromatography. Elute with water and follow by a mixtureof equal amount of 50% ethanol and ammonia water solution until theeluent became colourless. Concentrate the eluent and dry it to obtainthe sample (7).

Sample 8 was made with the method described below: Pulverize mulberryleaves and extract with 30% ethanol for 3 times (5 folds of the weightof the mulberry leaves, each time). Concentrate the combined extract andto the concentrate add ethanol to precipitate. Add water to theconcentrated supernatant and put it through a D101 macroporous resincolumn and collect the fluid. Elute with 3 folds of water and combinethe fluid and water eluent to obtain fraction 1. Continue to elute thecolumn with 60% ethanol (5 folds) to obtain fraction 2. Adjust thefraction 1 to pH4 and put it through a cation exchange column. Wash thecolumn with water until the eluent became colourless, Elute with 0.5 Nammonia solution (8 folds) and collect the eluent to obtain fraction 3.Concentrate and dry fractions 2 and 3, respectively to obtain the sample(8).

The solubility terms used in the Table are defined below:

-   -   “Easily soluble” 1 g sample will dissolve in less than 1 ml        water;    -   “Soluble” 1 g sample will dissolve in 10-30 ml water;    -   “Average” 1 g sample will dissolve in 30-100 ml water;

The above results demonstrated that the product of the present inventionis better than the current products in activity, content of the keyactive principle, colour and water solubility.

1. A plant extract obtained from Morus plant leaves which has an IC₅₀value of less than 90 μg/ml with respect to inhibition of α-glucosidaseI, comprising: 5-40% (w/w) total imino sugars, as measured by at leastone of quantitative High Performance Liquid Chromatography (HPLC) andliquid chromatography/mass spectrometry (LC-MS), the imino sugarsincluding at least one of 1-deoxynojirimycin (DNJ), fagomine andN-methyl-DNJ; and 20-70% (w/w) total amino acids, wherein the extract isat least one of pale yellow and nearly white and is easily soluble inwater.
 2. The plant extract as claimed in claim 1, wherein the IC₅₀value is 5-40 μg/ml with respect to inhibition of α-glucosidase I. 3.The plant extract as claimed in claim 1, wherein the total imino sugarsis 8-30% (w/w).
 4. The plant extract as claimed in claim 1, wherein thetotal imino sugar is 15-20% (w/w).
 5. The plant extract as claimed inclaim 1, wherein the imino sugars further include at least one of 1,4dideoxy-1,4-imino-D-arabinitol (DAB), 2-O-α-D-galactopyranosyl-DNJ(GAL-DNJ) and calystegin B.
 6. The plant extract as claimed in claim 1,wherein the total amino acids range from 30-60% (w/w).
 7. The plantextract as claimed in claim 6, wherein the total amino acids is 40-50%(w/w).
 8. The plant extract as claimed in claim 1, wherein the Morus isselected from a. Morus alba L., b. Morus alba var multicaulis L., c.Morus nigra, and d. Morus australis Poir.
 9. The plant extract asclaimed in claim 1, wherein said extract contains 1-20% (w/w) of DNJcalculated on the basis of the total weight of the extract.
 10. Theplant extract as claimed in claim 1, wherein the DNJ is included at aconcentration of 2-10% (w/w) calculated on the basis of the total weightof the extract.
 11. The plant extract as claimed in claim 1 wherein theDNJ is included at a concentration of 4-6% (w/w) calculated on the basisof the total weight of the extract.
 12. The plant extract as claimed inclaim 1, wherein the DNJ is included at a concentration of 1-3% (w/w)calculated on the basis of the total weight of the extract.
 13. Theplant extract as claimed in claim 1, further comprising a pH valuebetween 5.5-6.5 in a 1% water solution.
 14. The plant extract as claimedin claim 1, wherein a UV spectrum of the extract shows a maximumabsorption at 218 nm and 263 nm.
 15. A method for producing an extract,from Morus plant leaves, which has an IC₅₀ value to inhibitα-glucosidase I at a concentration of less than 90 μg/ml comprising thesteps of: a. Conducting a water or alcoholic extraction step from leafmaterial; b. Conducting a column chromatography purification step usinga strong acidic cation exchange resin, washing with water, and elutingwith an ammonia solution collecting the eluent and removing the ammoniatherefrom; and d. Concentrating and drying the extract.
 16. The methodas claimed in claim 15, wherein: a. prior to conducting the water oralcoholic extraction step, further comprising the step of drying theplant leaves into coarse powder, and wherein the extraction step isconducted with 5-18 folds of 0-40% of a low molecular weight alcohol perthe quantity of the Morus plant leaves (w/w), and repeated up to 5times, b. the step of conducting the column chromatography purificationstep includes washing the column with 1-2 folds of water of the columnvolume and discarding the wash, eluting the column with 2-8 folds of0.2-1.0N ammonia water solution of the column volume, at an elution rateof 1-3 folds of the volume of the column per hour, and collecting theeluent with a pH value of between 9.0 and 11.0, and d. the step ofconcentrating and drying the extract includes pulverizing the driedextract to pass through a mesh sieve. 17-19. (canceled)
 20. The methodas claimed in claim 15, wherein following the step of conducting thecolumn chromatography purification step, further comprising the step ofsubjecting the eluent to column chromatography using a macroporousabsorption resin and collecting the solution, wherein the column iseluted with a volume ratio between the solution and the column ofbetween 20:1 to 5:1.
 21. A cosmetic or medicament comprising a plantextract as claimed in claim 1 in combination with one or more skinlightening agents selected from the group consisting of: vitamin C andits derivatives, kojic acid, arbutin, diacetylboldin, azelaic acid,octadecenedioic acid, undecylenoylphenylalanine, liquorice extract, aloeextract, watercress extract, ascophyllum extract, hops extract,glutathione, ecdysone and ellagic acid.
 22. The cosmetic or medicamentas claimed in claim 21, wherein said vitamin C derivative is magnesiumL-ascorbyl-2-phosphate (VC-PMG).
 23. The cosmetic or medicament asclaimed in claim 21, wherein a ratio of the extract and the one or moreskin lightening agents is from 10:1 to 1:1. 24-25. (canceled)